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Biotin-Avidin System-Based Delivery Enhances the Therapeutic Performance of MSC-Derived Exosomes.

Exosomes (EXs) shed by mesenchymal stem cells (MSCs) are potent therapeutic agents that promote wound healing and regeneration, but when use … of GelMA. Biotin-modified MSC-EXs (Bio-EXs) were then synthesized using an in situ self-assembling biotinylation strategy, which provided sufficient binding sites for MSC-EX delivery with little effect on their cargo composition. Thereafter, Bio-EXs were immobilized in Bio-GelMA via streptavidin to generate Bio-GelMA@Bio-EX hydrogels. An in vitro analysis demo …

Direct enzyme-linked aptamer assay (DELAA) for diagnosis of toxoplasmosis by detection of SAG1 protein in mice and humans.

Toxoplasma gondii is a single-celled parasite commonly found in mammals and birds. Diagnosis of toxoplasmosis largely depends on measurement … as obtained from E.coli lysates by purification and identification with immunoblotting, followed by biotinylation. The selected aptamers were amplified by PCR and DNA sequencing. The results showed that the aptamer-2, with the highest affinity to n-SAG1 in the sera of animals with minimal difference in the four aptamer candidates, has a high specificity and sen …

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NO … hondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2• …

Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct.

Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates with a retrom … milar to AQP2 under 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Cell surface biotinylation assay showed that dDAVP-induced AQP2 translocation to the apical plasma membrane was unaffected after SNX27 knockdown in mpkCCD cells. In contrast, the dDAVP-induced AQP2 protein abundance was significantly attenuated without changes in AQP2 mRNA ex …

Contribution of NADPH oxidase to membrane CD38 internalization and activation in coronary arterial myocytes.

The CD38-ADP-ribosylcyclase-mediated Ca(2+) signaling pathway importantly contributes to the vasomotor response in different arteries. Altho … in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2·- product …

Deregulated genes in sporadic vestibular schwannomas.

OBJECTIVE: In search of genes associated with vestibular schwannoma tumorigenesis, this study examines the gene expression in human vestibular nerve versus vestibular schwannoma tissue samples using microarray technology. MATERIAL AND METHODS: RNA was extracted from 3 vestibular nerves (serving as control) and 16 solid, sporadic vestibular schwannomas. RNA (5 microg) was used in the labeling and biotinylation protocol to produce cRNA, which was hybridized to Affymetrix HG-U133A arrays. Data were imported into dChip v.1.3 and normalized using invariant set normalization. Differentially express…

Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

Mutations in the voltage-gated K(+) channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we p … 255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, …

Effect of the cGMP pathway on AQP2 expression and translocation: potential implications for nephrogenic diabetes insipidus.

BACKGROUND: Arginine vasopressin (AVP) binding to the V2 receptor (V2R) in renal collecting duct principal cells induces a cAMP signalling c … ed with ANP, l-arginine and 8-Br-cGMP for 2 h and subjected to immunocytochemistry and cell surface biotinylation assays to examine their effect on AQP2 translocation. To test the effect of cGMP pathway activators on AQP2 expression, the mpkCCD cells were treated with dDAVP, ANP and l-arginine for 4 days, or with 8-Br-cGMP for the last day. AQP2 protein levels …

Распространение и сигнализация TREM2/DAP12, рецепторная система мутировала в человеческом полициклическом липонимбранном остеодеспласе с склерозным лейкоэнцефалопатическим слабоумием.

Вместе со своим адапторным протеином, адапторным белком 12 кДа, также известным как CARAP и TYROBP (DAP12), инициирующий рецептор, выраженный в миэлоидных клетках 2 (TREM2), является стимулирующим мембранным рецептором иммуноглобулина/литинового суперсемейства, хорошо известного в миэлоидных клетках. Однако в организме человека мутации функций TREM2/DAP12 оставляют миелоидные клетки нетронутыми, но вызывают автосомную рентгеновскую болезнь, характеризующуюся, вместе с костными костными кистами, спектром патологических повреждений в коре, таламусе и базальной ганглии с клиническими симптомами п

Совпадение в аннотации оригинала
Together with its adaptor protein, the adaptor protein of 12 kDa also known as KARAP and TYROBP (DAP12), triggering receptor expressed in my … cantly and transiently (60 min) when cells are stimulated by ionomycin, as revealed by both surface biotinylation and surface immunolabeling. Our results provide the first information about the expression, distribution (mostly intracellular) and functioning of TREM2/DAP12 system in nerve cells, a necessary step in the understanding of the cellular mechanisms af …

Nongenomic effect of aldosterone on Na+,K+-adenosine triphosphatase in arterial vessels.

Aldosterone increases Na(+),K(+)-adenosine triphophatase (Na(+),K(+)-ATPase) pump activity and abundance under chronic conditions in several … ect of aldosterone was inhibited by disrupters of microtubule (colchicine). Plasma membrane protein biotinylation of aortic segments and Western blot indicated a diminished presence of catalytic isoforms of Na(+),K(+)-ATPase on the cell surface. Our findings indicate that aldosterone has a nongenomic effect on the Na(+),K(+)-ATPase of vascular tissue. This effe …

LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species.

Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protecti … ated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and con …

Dynamic properties of nucleated microtubules: GTP utilisation in the subcritical concentration regime.

Microtubule assembly kinetics have been studied quantitatively under solution conditions supporting microtubule dynamic instability. Purified GTP-tubulin (Tu-GTP) and covalently cross-linked short microtubule seeds (EGS-seeds; Koshland et al. (1988) Nature 331, 499) were used with and without biotinylation. Under sub-critical concentration conditions ([Tu-GTP] < 5.3 microM), significant microtubule growth of limited length was observed on a proportion of the EGS-seeds by immuno-electron microscopy. A sensitive fluorescence assay for microtubule GDP production was developed for parallel assessm…

Characterisation of the purified human sodium/iodide symporter reveals that the protein is mainly present in a dimeric form and permits the detailed study of a native C-terminal fragment.

The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domai

Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain).

The ICR-derived glomerulonephritis (ICGN) mice consist of heterozygous and homozygous groups and are considered to be a good model for human idiopathic nephrotic syndrome. To reveal changes in cell-surface carbohydrate construction, 24 lectins were applied to kidney sections of 10-, 30- and 50-week-old male heterozygous and homozygous ICGN mice and age-matched male ICR mice. Bandeiraea simplicifolia lectin-I (BSL-I), which specifically binds to alpha-D-galactopyranosyl groups, showed positive staining in the glomeruli of ICGN mice, but not in those of ICR mice. Positive BSL-I staining was obse

Hyaluronic acid suppresses fibronectin fragment mediated cartilage chondrolysis: I. In vitro.

A commercial preparation of 800-kDa hyaluronic acid (HA), (ARTZ from Seikagaku, Inc.), has been used as a therapeutic intervention in the treatment of osteoarthritis (OA). We tested the effect of this HA form, HA/800, in an in vitro cartilage chondrolytic system in which a specific amino-terminal 29-kDa fragment of fibronectin (Fn-f) penetrates cartilage tissue to activate chondrocytes to amplify two major chondrolytic activities: suppression of proteoglycan (PG) synthesis and induction of matrix metalloproteinases. We report that HA/800 did not block damage by Fn-f in serum free cartilage cul

Uncovering essential RNA components of fusion oncoprotein condensates using bicon-seq.

Fusion oncoproteins drive aggressive leukemias by mediating aberrant gene expression within specialized biomolecular condensates that contain various biomolecules. While RNA is known to influence condensate properties, the RNA constituents of fusion protein-containing onco-condensates remain poorly defined. Here, we use bicon-seq (biotinylated-isoxazole-mediated precipitation of condensome and RNA sequencing) to globally profile condensate-associated RNAs from cellular samples. Using experimental perturbation of cytoplasmic RNA-containing condensates, we demonstrated that bicon-seq accurately

Targeting the ZDHHC9-mediated STAT1 palmitoylation-phosphorylation conversion inhibits gastric cancer progression.

BACKGROUND: Palmitoyl transferases ZDHHCs and its mediated protein palmitoylation in several malignant tumors have been reported. However, the role in gastric cancer (GC) has not yet been fully elucidated. METHODS: Various experimental techniques were employed to investigate the role of ZDHHCs and related protein palmitoylation in GC, mainly including acyl-biotinyl exchange assay, co-immunoprecipitation, immunohistochemistry, western blot, immunofluorescence, and confocal imaging. RESULTS: We identified widespread protein palmitoylation in GC, and the palmitoylation inhibitor 2-bromopalmitat

Engineering viral vectors with CD200 enhances anti-inflammatory and phagocytosis resistance.

CD200, an immunoregulatory glycoprotein of the immunoglobulin superfamily, suppresses inflammatory signaling by engaging its receptor CD200R, which is predominantly expressed on myeloid cells. To enhance the immune-evading properties of viral vectors, we engineered lentiviral particles displaying the CD200 ectodomain (CD200ED) to exploit anti-inflammatory response and phagocytosis resistance. A fusion gene encoding the mouse CD200 ectodomain and core streptavidin (CD200ED-coreSA) was cloned into the pET-30a(+) plasmid, expressed in E. coli Lemo21(DE3), and purified via immobilized metal affini

The Dibasics of M13 phage display: examining the substrate specificity of Escherichia coli outer membrane proteases.

Bacteriophage M13 substrate libraries are a useful tool to discover novel protease substrates. In this study, we have mapped 7-mer protease substrates cleaved by native Escherichia coli proteases during phage outgrowth. Cleavage was monitored by the loss of a biotinylated AviTag at the N-terminus of the recombinant M13 minor coat protein, protein III. Using this method, we isolated 120 phage-displayed substrates that contain dibasic amino acid motifs (Lys-Lys, Arg-Arg, Lys-Arg, and Arg-Lys), of which the Lys-Lys motif has not been previously described using substrate phage. Since dibasic motif

Fluorescent Detection of Aqueous N-Nitrosodimethylamine via Photochemical Transformation and Affinity Capture.

N-Nitrosodimethylamine (NDMA) is a water-soluble carcinogen typically quantified using mass spectrometry coupled with chromatographic separation, which requires extensive sample preparation and sophisticated instrumentation. Here, we report a fluorescence-based detection strategy for NDMA in aqueous media that replaces column-based separation with affinity capture. Photochemical transformation of NDMA, followed by reaction with a biotinylated polymeric reagent, generates a biotin-labeled fluorescent product that can be selectively enriched through streptavidin-biotin interaction, enabling effe